Project 3 ABSTRACT Project 3 aims to develop a tiered translational in vitro-to-in vivo experimental testing strategy for evaluating the inter-tissue and inter-individual variability in responses to complex environmental mixtures. This goal is a critical part of the overall strategy of the Texas A&M University Superfund Research Center to characterize and manage the human health risks associated with exposure to environmental emergency-mobilized hazardous substances through the development of tools that can be used by first responders, the impacted communities, and the government bodies involved in site management and cleanup. We have recently demonstrated that organotypic and population-based experimental models for toxicology can enable not only a more rapid identification of chemical hazards, but also serve as the opportunity to test real-life exposures. Our overall hypothesis is that a tiered, risk-based strategy for safety evaluation consisting of human and mouse organotypic in vitro cultures and Collaborative Cross mouse strains, combined with a population-based reverse toxicokinetics, is a sensible ?fit-for-purpose? approach to characterizing hazards of complex mixtures from contamination events during environmental emergencies. First, we will develop a multi-tissue ?biological read- across? approach for complex environmental mixtures using high-content/-throughput assays with human induced pluripotent stem cells (iPSC). Data from high-content screening and high-throughput genomic analyses with human iPSC-derived organotypic cultures (hepatocytes, cardiomyocytes, endothelial cells, macrophages, neurons, etc.) will be used to categorize the effects of mixtures with respect to the magnitude and tissue-specificity of the hazard. Second, we will develop a population-based in vitro-in vivo approach in mice to characterize inter-tissue and inter-individual variability in responses to complex environmental mixtures, including the Galveston Bay/Houston Ship Channel site samples that will be collected in Project 1. We will use the Collaborative Cross, a panel of genetically diverse mouse strains that model human population variability, to establish a library of mouse iPSC-derived embryoid bodies that encompass diverse cell lineages. This population-based in vitro model will be used in toxicity screening of chemicals and mixtures. Third, we will develop a high-throughput reverse toxicokinetics modeling approach for in vitro-to-in vivo extrapolation (IVIVE) of quantitative estimates of hazard for complex environmental mixtures. In partnership with the Exposure Science Core, we will utilize modern untargeted metabolomics methods to deconvolute complex mixtures into toxicokinetically-similar components on which high-throughput IVIVE can be performed. Finally, we will demonstrate the utility of our biological read-across approach for quantitative estimation of hazard for complex environmental mixtures. We will partner with the Data Science and Decision Science Cores to show how in vitro toxicity and transcriptomic data, properly adjusted for toxicokinetics with IVIVE, can identify biological analogues to complex mixtures from a library of reference compounds. These data and methods will be translated into tools that can protect human health during/after environmental disaster emergencies.